Experimental studies on proteins using NMR relaxation and hydrogen exchange have unequivocally established the existence of states that are conformational excursions of the canonical high resolution structure, even under native conditions. Despite knowledge of their existence, however, little is known of the nature and energetics of these states. The importance of understanding the structural and energetic details of the conformational states that exist under native conditions cannot be overstated. As the observed biological activity of a protein is the energy (or Boltzmann)-weighted contribution of the component microstates in the ensemble, knowledge of the structural and thermodynamic features of these states is a prerequisite to a molecular-level understanding of protein function. An experimental strategy has been developed that takes advantage of the thermodynamic linkage between stability and binding affinity. According to this linkage scheme, by monitoring the effects of ALA to GLY mutations on the observed binding affinity (using isothermal titration calorimetry) and stability (using hydrogen-deuterium exchange), it is possible to directly determine;1) the similarity between fluctuations and 'local unfolding;'2) the quantitative impact of fluctuation on binding;3) the effect of urea, osmolytes, temperature, and pH on this behavior;and 4) the variability of the results obtained at different sites within a particular loop, and at different loops within the two model proteins. The strategy is applied to the analysis of several loops in two model proteins;the C-SH3 domain of SEM5, and E. Coli. dihydrofolate reductase (DHFR). The studies described herein represent a unique strategy for elucidating the structure and energy of the conformational variants of the canonical structure, which are populated even under native conditions.